Influenza is the leading cause of death in humans due to a respiratory virus. Common symptoms include fever, sore throat, shortness of breath, and muscle soreness, among others. During flu season, influenza viruses infect 10-20% of the population worldwide, leading to 250-500,000 deaths annually.
Influenza viruses are classified into types A, B, or C, based on the nucleoproteins and matrix protein antigens present. Influenza type A viruses may be further divided into subtypes according to the combination of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins presented. HA governs the ability of the virus to bind to and penetrate the host cell. NA removes terminal sialic acid residues from glycan chains on host cell and viral surface proteins, which prevents viral aggregation and facilitates virus mobility. Currently, 16 HA (H1-H16) and 9 NA (N1-N9) subtypes are recognized. Each type A influenza virus presents one type of HA and one type of NA glycoprotein. Generally, each subtype exhibits species specificity; for example, all HA and NA subtypes are known to infect birds, while only subtypes H1, H2, H3, H5, N1 and N2 have been shown to infect humans. Influenza viruses comprising H5 and H7 are considered the most highly pathogenic forms of influenza A viruses, and are most likely to cause future pandemics.
Influenza pandemics are usually caused by highly transmittable and virulent influenza viruses, and can lead to elevated levels of illness and death globally. The emergence of new influenza A subtypes resulted in 4 major pandemics in the 20th century. The Spanish flu, caused by an H1N1 virus, in 1918-1919 led to the deaths of over 50 million people worldwide between 1917 and 1920. The risk of the emergence of a new subtype, or of the transmission to humans of a subtype endemic in animals, is always present. Of particular concern is a highly virulent form of avian influenza (also called “bird flu”), outbreaks of which have been reported in several countries around the world. In many cases, this bird flu can result in mortality rates approaching 100% within 48 hours. The spread of the avian influenza virus (H5N1), first identified in Hong Kong in 1997, to other Asian countries and Europe has been postulated to be linked to the migratory patterns of wild birds.
There is increasing concern that the virus may become highly infectious for humans. The major problem for human health is the fact that influenza viruses are antigenically unstable, that is, they mutate rapidly. Should the avian influenza virus come into contact with human viruses, genetic reassortment of the avian virus could result in a highly pathogenic influenza virus that could causes severe disease or death in humans. Furthermore, such mutation could result in an influenza virus that is easily transmitted in humans.
The current method of combating influenza in humans is by annual vaccination. Each year, the World Health Organization selects 3 viral strains for inclusion in the annual influenza vaccine, which is produced in fertilized eggs. However, the number of vaccine doses produced each year is not sufficient to vaccinate the world's population. For example, Canada and the United-States obtain enough vaccines doses to immunize about one third of their population, while only 17% of the population of the European Union can be vaccinated. It is evident that current worldwide production of influenza vaccine would be insufficient in the face of a worldwide flu pandemic. Therefore, governments and private industry alike have turned their attention to the productions of effective influenza vaccines.
As previously mentioned, the current method of obtaining influenza virus vaccines is by production in fertilized eggs. The virus is cultured in fertilized eggs, followed by inactivation of the virus and purification of viral glycoproteins. While this method maintains the antigenic epitope and post-translational modifications, there are a number of drawbacks including the risk of contamination due to the use of whole virus and variable yields depending on virus strain. Sub-optimal levels of protection may result from genetic heterogeneity in the virus due to its introduction into eggs. Other disadvantages includes extensive planning for obtaining eggs, contamination risks due to chemicals used in purification, and long production times. Also, persons hypersensitive to egg proteins may not be eligible candidates for receiving the vaccine.
To avoid the use of eggs, influenza viruses have also been produced in mammalian cell culture, for example in MDCK or PERC.6 cells, or the like. Another approach is reverse genetics, in which viruses are produced by cell transformation with viral genes. These methods, however, also requires the use of whole virus as well as elaborate methods and specific culture environments.
The use of viral DNA as a vaccine has been explored. In this technology, protection is obtained by expression of viral antigens in human cells; the antigens are then recognized as foreign antigen, which leads to an specific antibody response. However, there exists the risk of oncogene activation from introduction of DNA into determinant portion of human cell genome—a significant drawback.
Vaccines comprising recombinant viral antigens expressed in viral DNA transformed insect or plant cells have also been prepared by Dow Agroscience (see, for example WO 2004/098530). While the risk associated with the use of live virus is avoided and the production process is shorter, protein conformation and post-translational modifications are affected. The scale-up and purification steps are also relatively complex as the antigens are associated with cellular membranes. In addition, the dose of baculovirus-recombinant HA required for effective immunization in animals is 10-fold higher than that of natural HA produced in fertilized eggs. In both cases, the levels of viral antigen expression are low.
In an effort to avoid the difficulties associated with purification of membrane proteins, Huang et al (2001, Vaccine, 19:2163-2171) replaced the transmembrane domain and the cytoplasmic tail of the measles HA with an ER retention signal. The resulting HA protein was produced in tobacco plant cells for the development of oral vaccine (edible vaccine). The HA expressed is not as strongly retained in the ER as it is with the transmembrane domain, thus simplifying the purification procedure. However, the natural trimeric form of HA cannot be formed in those conditions, which can affect the immunogenicity of the recombinant protein.
Saelens et al (1999, Eur. J. Biochm, 260:166-175) expressed an HA gene lacking the transmembrane domain in yeast (Pichia pastoris), leading to the secretion of monomeric HA. This form, however, was less immunogenic than the trimeric HA.
In order to protect the world population from influenza and to stave off future pandemics, vaccine manufacturers will need to develop effective, rapid methods producing vaccine doses. The current use of fertilized eggs to produce vaccines is insufficient and involves a lengthy process. Recombinant technologies offer promising approaches to the production of influenza antigens. However, the production of hemagglutinin has been limited to membrane-associated protein, which involves complex extraction processes with low yields, or to poorly-immunogenic soluble proteins.